ES2742179T3 - Pharmaceutical compositions comprising orexin-1 OX1R receptor agonists for the treatment of inflammatory bowel diseases - Google Patents

Abstract

At least one OX1R agonist for use in a method of treating an inflammatory bowel disease in a subject in need, where the OX1R agonist is a polypeptide that is at least 70% identical with SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

Description

DESCRIPTION

Pharmaceutical compositions comprising orexin-1 OX1R receptor agonists for the treatment of inflammatory bowel diseases

Field of the Invention

Pharmaceutical methods and compositions for the treatment of inflammatory bowel diseases are described herein.

Background of the invention

Inflammatory bowel disease (IBD), including Crohn's disease (CD) and UC affects approximately 1.5 million Europeans, and its maximum onset is in people 15 to 30 years of age. The incidence of IBD continues to increase in developing countries. In France, 64,399 patients with UC have been estimated from the records of the 2008 health security agency. CU involves the rectum and can affect part of the colon or the entire colon (pancolitis) in an uninterrupted pattern. Changes in diet, antibiotic use, environmental risk factors and intestinal microflora are involved in the pathogenesis of IBD and have contributed to the higher prevalence of IBD over the past century.

Despite the enormous advances in the understanding by the inventors of the pathobiology of IBD, current treatments are not efficient to control the disease in the long term. In fact, 15 to 25% of patients with UC are still operated (total colectomy) and the most potent treatments (anti-TNF antibodies) provide complete remission per year of treatment in only 30% of patients enrolled in trials. clinical. In addition, UC increases the risk of colorectal cancer (18% at 30 years). Recently, the estimated annual direct medical costs per CU patient ranged from $ 6217 to $ 11477 in the US. UU. and between € 8949 and € 10395 in Europe. Hospitalizations accounted for 41-55% of direct medical costs. Indirect costs accounted for approximately one third of total costs in the United States and 54-68% in Europe. The total economic burden of the UC was estimated at $ 8.1-14.9 billion annually in the US. UU. and at € 12.5 29.1 billion in Europe. Total direct costs were $ 3.4-8.6 billion in the United States and € 5.4-12.6 billion in Europe. Direct costs, hospitalizations and surgeries increased with the worsening of the severity of the disease. CU is an expensive disease and hospitalizations contribute significantly to direct medical costs, and indirect costs are considerable. Better medical control of the disease is required. Until now, current treatments are used empirically in CU. They mainly target the immune system and inflammation (aminosalicylates, steroids and immunomodulators), while new treatments that target epithelial abnormalities (i.e., ER stress, oxidative stress) are likely to be more effective (and better tolerated) , to maintain prolonged remission or even to cure UC. Therefore, drugs that prevent primary epithelial defects are mandatory to better control UC, improve the patient's quality of life, avoid terminal complications and significantly reduce the costs of UC care.

The orexins (orexin-A and orexin-B) are hypothalamic peptides involved in sleep / wake control that interact with two subtypes of GPCR class A, OX1R and OX2R. Activation of these receptors by orexins normally induces transient calcium cellular agents through Gq-dependent and independent pathways. Recently it has been shown that OX1R was highly ectopically expressed in digestive cancer cell lines derived from colon, pancreas and liver cancers in which orexins: 1) induce a strong mitochondrial apoptosis; 2) induce a strong inhibition of tumor growth in nude mice xenografted with cancer cells. These effects were mediated by an original mechanism that involves: i) the presence of two ITIM sequences (tyrosine immunoreceptor inhibitory motif) in OX1R that are phosphorylated in tyrosine in the presence of orexins; ii) the recruitment and activation of tyrosine phosphatase SHP-2, which is responsible for mitochondrial apoptosis involving the release of cytochrome c from mitochondria to cytosol and activation of caspase-3 and caspase-7. However, the involvement of orexins in the treatment of IBD has never been investigated.

Summary

As defined in the claims, the present invention relates to at least one OX1R agonist for use in a method of treating an inflammatory bowel disease in a subject in need thereof, in which the OX1R agonist is a polypeptide having At least 70% identity with SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

Detailed description

The inventors have shown that OX1R was detected in human epithelia of inflammatory bowel disease (IBD), including Crohn's disease and ulcerative colitis (UC). Based on these observations, the effect of Orexin A (OxA) on acute inflammation in mice treated with Sodium Dextran Sulfate (DSS) has been investigated and it has been shown that OxA treatment of mice treated with DSS improves the Index of Disease Activity (ICD) and also the histological aspect of the colon epithelium. In addition, OxA reduces the secretion of "proinflammatory" cytokines such as TNFa, IL6, homologue of IL8 and IL1B in colon extracts of mice with DSS-induced colitis. The recent development of a new model of spontaneous mouse type UC (EXCY2 mice, document WO / 2012/140516) is ideal for studying the effect of OxA on spontaneous colitis. The inventors have shown that treatment with OxA protected from severe colitis in EXCY2 mice 10 weeks old. In fact, EXCY2 mice treated with OxA exhibited a normal colonic mucosa compared to vehicle-treated EXCY2 mice. In addition, the OxA / OXIR system induced two major deregulated pathways involved in the onset of colitis, that is, oxidative stress and ER stress, both in EXCY2 mice and in Caco2 cells. These results indicate that OX1R agonists are suitable for the treatment of inflammatory bowel disease.

Accordingly, a method of treating an inflammatory bowel disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of at least one OX1R agonist, is described herein.

As used herein, the term "subject" denotes a mammal, such as a rodent, a feline, a canine and a primate. Preferably, a subject as described herein is a human being.

As used herein, the term "inflammatory bowel disease" has its general meaning in the art and refers to any inflammatory disease that affects the intestine. The expression includes, among others, ulcerative colitis, Crohn's disease, especially Crohn's disease in a state that specifically affects the colon with or without ileitis, microscopic colitis (lymphocytic colitis and collagen colitis), infectious colitis caused by bacteria or viruses, Radiation colitis, ischemic colitis, pediatric colitis, indeterminate colitis and functional intestinal disorders (symptoms described without obvious anatomical abnormalities). In some embodiments, the method described herein is particularly suitable for the treatment of ulcerative colitis.

As used herein, "treatment" or "treat" is an approach to obtain beneficial or desired results that include clinical results. For the purposes of this invention, beneficial or desired clinical results include, among others, one or more of the following: relieve one or more symptoms resulting from the disease, decrease the extent of the disease, stabilize the disease (e.g., prevent or delay the worsening of the disease), prevent or delay the spread of the disease, prevent or delay the recurrence of the disease, delay or delay the progression of the disease, improve the disease status, provide a remission (partial or total ) of the disease, decreasing the dose of one or more medications necessary to treat the disease, delaying the progression of the disease, increasing the quality of life and / or prolonging survival. The term "treatment" encompasses prophylactic treatment. As used herein, the term "prevent" refers to the reduction of the risk of acquiring or developing a given condition, or the reduction or inhibition of recurrence or said condition in a subject who is not sick, but who has been or it may be near a subject with the disease.

As used herein, the term "OX1R" has its general meaning in the art and refers to the OX1R receptor spanning 7 transmembrane domains for orexins. An exemplary amino acid sequence of OX1R is shown as SEQ ID NO: 1.

SEQ ID NO: 1: human orexin receptor-1 OX1R

1 mepsatpgaq mgvppgsrep spvppdyede flrylwrdyl ypkqyewvli aayvavfvva

61 Ivgntlvcla vwmhhmrtv tnyfivnlsl advlvtaicl pasllvditc swlfghalck

121 vipylqavsv svavltlsfí aldrwyaich pllfkstarr argsilgiwa vslaimvpqa

181 avmecssvlp elanrtrlfs vcderwaddl ypkiyhscff ivtylaplgl mamayfqifr

241 klwgrqipgt tsalvmwkr psdqlgdleq glsgcpqprg raflaevkqm rarrktakml

201 m wllvfalc ylpisvlnvl krvfgmfrqa sdreavyacf tfshwlvyan saanpiiynf

261 lsgkfrcqfk aafscclpgl gpcgslkaps prssashksl slqsrcsisk isehw ltsv

421 ttvlp

Therefore, as used herein, the term "OX1R agonist" refers to any natural compound or that cannot bind to OX1R and promotes the activity of OXIR. In particular, an easy method to determine if a compound is an OX1R agonist is to determine if the candidate can induce a transient release of calcium in a cell that expresses OX1R. In some other aspects, a compound is an OX1R agonist when it induces apoptosis of cancerous cell lines that express OX1R (eg, colorectal cancer cell lines). In fact, OX1R promotes apoptosis in cancer cell lines through a mechanism that is not related to the activation of Gq-mediated phospholipase C and agents transient calcium cells. The orexins do in fact induce tyrosine phosphorylation of 2 motifs based on tyrosine in OX1R, ITIM and ITSM, resulting in the recruitment of phosphotyrosine phosphatase SHP-2, whose activation is responsible for mitochondrial apoptosis (Voisin T, Firar A , Rouyer-Fessard C, Gratio V, Laburthe M. “A hallmark of immunoreceptor, the tyrosine-based inhibitory motif ITIM, is present in the G protein-coupled receptor OX1R for orexins and drives apoptosis: a novel mechanism.” PHASEB J. 2008 Jun; 22 (6): 1993-2002; The Firar A, Voisin T, Rouyer-Fessard C, Ostuni MA, Couvineau A, Laburthe M. “Discovery of a functional immunoreceptor tyrosine-based switch motif in a 7-transmembrane- spanning receptor: role in the orexin receptor OX1R-driven apoptosis. ”FASEB J. 2009 Dec; 23 (12): 4069-80. doi: 10.1096 / fj.09-131367. Epub August 6, 2009) of the activation of signal transduction pathways that involve the recruitment of SHP-2 and the induction of apoptosis of c cell, regardless of the temporary release of calcium.

In one embodiment, the OX1R agonist is a polypeptide. In a particular embodiment, the polypeptide is a functional equivalent of Orexin-A or Orexin-B.

As used herein, the term "Orexin-A" has its general meaning in the art and refers to the amino acid sequence as shown in SEQ ID NO: 2.

Orexin-A: ^p eplpdccrqk tcscrlyell hgagnhaagi ltlx (SEQ ID NO: 2)

^p e means 2-pyrrolidone-5-carboxylic acid (Xaa: pyrrolidone-carboxylic acid).

As used herein, the term "orexin-B" has its general meaning in the art and refers to the amino acid sequence shown in SEQ ID NO: 3.

Orexin-B: fsgppglqgr lqrllqasgn haagiltm (SEQ ID NO: 3) or glqgr lqrllqasgn haagiltm (SEQ ID NO: 4).

As used herein, a "functional equivalent of orexin" is a polypeptide that is capable of binding to OX1R, thereby promoting an OX1R activity according to the invention. The term "functional equivalent" includes fragments, mutants and muteins of Orexin-A and Orexin-B. The term "functionally equivalent" thus includes any equivalent of orexins (ie, Orexin-A or Orexin-B) obtained by altering the amino acid sequence, for example, by one or more amino acid deletions, substitutions or mode additions. that the protein analog retain the ability to bind to OX1R and promote an activity of OX1R according to the invention (eg, apoptosis of the cancer cell). Amino acid substitutions can be made, for example, by timely mutation of the DNA encoding the amino acid sequence.

In some embodiments, the functional equivalent has at least 70% identity with respect to the corresponding protein. According to the invention, a first amino acid sequence that has at least 70% identity with a second amino acid sequence means that the first sequence has 70; 71; 72; 73; 74; 75; 76; 77; 78; 79; 80; 81; 82; 83; 84; 85; 86; 87; 88; 89; 90; 91; 92; 93; 94; 95; 96; 97; 98; or 99% identity with the second amino acid sequence and that preserves the biological properties of said second amino acid sequence. The identity of the amino acid sequence is preferably determined using a suitable sequence alignment algorithm and predetermined parameters, such as BLAST P (Karlin and Altschul, 1990).

The term "a functionally equivalent fragment," as used herein, can also mean any fragment or set of Orexin fragments that binds OX1R and promotes the activity of OX1R according to the invention. Accordingly, the present invention provides a polypeptide comprising consecutive amino acids having a sequence corresponding to the sequence of at least one part of Orexin-A or Orexin-B, whose part binds to OX1R and promotes the activity of OX1R according with the invention

The polypeptides described herein can be produced by any suitable means, as will be apparent to those skilled in the art. In order to produce sufficient amounts of polypeptides or functional equivalents thereof for use in accordance with the present invention, expression can be conveniently achieved by culturing under appropriate conditions recombinant host cells containing the polypeptide described herein. In particular, the polypeptide is produced by recombinant means, by expression of a coding nucleic acid molecule. Cloning and expression systems of a polypeptide in a variety of different host cells are common. When expressed recombinantly, the polypeptide is generated in particular by its expression from a coding nucleic acid in a host cell. Any host cell can be used, depending on the individual requirements of a particular system. Suitable host cells include mammalian bacterial cells, plant cells, yeasts and baculovirus systems. Mammalian cell lines available in the art for the expression of a heterologous polypeptide include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells and many others. Bacteria are also preferred hosts for recombinant protein production, due to the ease with which bacteria can be manipulated and cultured. A common and preferred bacterial host is E. coli.

In some embodiments, the polypeptide described herein is an immunoadhesin.

As used herein, the term "immunoadhesin" designates antibody-like molecules that combine the binding specificity of a heterologous protein (an "adhesin" that is capable of binding to OX1R) with the effector functions of the constant domains of immunoglobulin Structurally, immunoadhesins comprise a fusion of an amino acid sequence with the desired binding specificity to OX1R (ie, it is "heterologous"), and a sequence of the immunoglobulin constant domain. The adhesin part of an immunoadhesin molecule is usually a contiguous amino acid sequence that comprises at least the binding site for OX1R. In one embodiment, adhesin comprises polypeptides characterized by SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4. The immunoglobulin constant domain sequence in the immunoadhesin can be obtained from any immunoglobulin, such as the IgG-1, IgG-2, IgG-3 or IgG-4, IgA subtypes (including IgA-1 and IgA-2), IgE , IgD or IgM.

The immunoglobulin sequence, although not necessarily, is usually a constant domain of immunoglobulin (Fc region). Immunoadhesins can possess many of the valuable chemical and biological properties of human antibodies. Since immunoadhesins can be constructed from a sequence of human proteins with the desired specificity linked to a suitable human immunoglobulin hinge and a constant domain sequence (Fc), the binding specificity of interest can be achieved using completely human components. . These immunoadhesins are minimally immunogenic for the patient and are safe for chronic or repeated use.

In one embodiment, the Fc region is an Fc region of native sequence. In one embodiment, the Fc region is a variant Fc region. In yet another embodiment, the Fc region is a functional Fc region. As used herein, the term "Fc region" is used to define a C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain may vary, the region of the human IgG Fc heavy chain is generally defined to extend from an amino acid residue at the Cys226 position, or from Pro230, to the carboxyl end of the same. The adhesion portion and part of the immunoadhesin immunoglobulin sequence may be linked by a minimum linker. The immunoglobulin sequence typically, but not necessarily, is a constant domain of immunoglobulin. The rest of the immunoglobulin in the chimeras described herein can be obtained from the subtypes IgG1, IgG2, IgG3 or IgG4, IgA, IgE, IgD or IgM, but usually IgG1 or IgG3.

The polypeptides described herein, fragments thereof and fusion proteins (for example, immunoadhesin) according to the invention may exhibit post-translational modifications, including, but not limited to glycosylations, (eg, N-linked glycosylations or O), myristylations, palmylations, acetylations and phosphorylations (for example, serine / threonine or tyrosine).

In specific embodiments, it is contemplated that the polypeptides used in the therapeutic methods described herein may be modified in order to improve their therapeutic efficacy. Said modification of the therapeutic compounds can be used to decrease toxicity, increase circulatory time or modify biodistribution. For example, the toxicity of potentially important therapeutic compounds can be significantly reduced by their combination with a variety of drug carriers that modify biodistribution. For example, the addition of dipeptides can improve the penetration of a circulating agent into the eye through the blood-retina barrier through the use of endogenous transporters.

A strategy to improve the viability of medicines is the use of water soluble polymers. It has been shown that various water-soluble polymers modify biodistribution, improve the mode of cellular absorption, change permeability through physiological barriers; and modify the rate of clearance in the body. To achieve a sustained recognition or release effect, water soluble polymers containing drug residues have been synthesized as terminal groups, as part of the structure or as pending groups in the polymer chain.

Polyethylene glycol (PEG) has been widely used as a vehicle for drugs, given its high degree of biocompatibility and ease of modification. It has been shown that binding to various medications, proteins and liposomes improves residence time and decreases toxicity. PEG can be coupled to active agents through hydroxyl groups at the ends of the chain and through other chemical methods; however, the PEG itself is limited to a maximum of two active agents per molecule. In a different approach, copolymers of PEG and amino acids were explored as new biomaterials that would retain the biocompatibility properties of PEG, but would have the added advantage of providing numerous binding points per molecule (providing a greater pharmaceutical load), being able to be synthetically designed to Adapt to a variety of applications.

Those skilled in the art know about PEGylation techniques for effective drug modification. For example, polymers have been used for the administration of medicaments consisting of alternative PEG polymers and trifunctional monomers such as lysine by VectraMed (Plainsboro, NJ). PEG chains (typically 2000 daltons or less) are linked to lysine a- and e-amino groups through stable urethane bonds. Such copolymers retain the desirable properties of PEG, while providing reactive dependent groups (lysine carboxylic acid groups) at strictly controlled intervals and predetermined along the polymer chain. Reactive dependent groups can be used for derivation, crosslinking or conjugation with other molecules. These polymers are useful for producing stable and long-circulating drugs by varying the molecular weight of the polymer, the molecular weight of the PEG segments and the cleavage link between the drug and the polymer. The molecular weight of the PEG segments affects the spacing of the link / drug group complex and the amount of drug per molecular weight of the conjugate (the smaller PEG segments provide a greater drug load). In general, increasing the overall molecular weight of the block copolymer conjugate will increase the circulatory half-life of the conjugate. However, the conjugate must be easily degradable or have a molecular weight below the filtration threshold by glomular limitation (for example, less than 60 kDa).

In addition, it is important for the polymeric structure to maintain the circulatory half-life and biodistribution, the links can be used to maintain the therapeutic agent in the form of a pro-drug until it is released from the polymeric structure by a specific trigger, usually the Enzymatic activity in the target tissue. For example, this type of administration of tissue activated drugs is particularly useful when delivery to a specific biodistribution site is required and the therapeutic agent is released at or near the pathology site. The linker group libraries for use in the administration of activated drugs are known to those skilled in the art and can be based on enzyme kinetics, the prevalence of the active enzyme and the specificity of cleavage of the disease-specific enzymes selected. . Such linkers can be used in the modification of the protein or protein fragment described herein for therapeutic administration.

The terms "administer" or "administration" refer to the act of injecting or physically administering a substance as it exists outside the body (for example, an OX1R agonist described herein) in the subject, such as, for example, by the mucosal, intradermal, intravenous, subcutaneous, intramuscular and / or any other method of physical administration described herein or known in the art. When a disease or symptom is being treated, the administration of the substance usually occurs after the onset of the disease or its symptoms. When a disease or its symptoms are prevented, the administration of the substance usually occurs before the onset of the disease or its symptoms.

In some embodiments, the OX1R agonist described herein is administered to the subject with a therapeutically effective amount.

A "therapeutically effective amount" means a sufficient amount of OX1R to treat inflammatory bowel disease in a reasonable benefit / risk ratio applicable to any medical treatment. It will be understood that the total daily use of the compounds and compositions described in this document will be decided by the attending physician within the scope of a sound medical judgment. The level of therapeutically effective dose specific to any particular subject will depend on a variety of factors, including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed, age, body weight, general health, sex and diet of the subject; the time of administration, the route of administration and the rate of excretion of the specific compound employed; the duration of treatment; medicines used in combination or coincident with the specific polypeptide used; and as usual factors in the medical arts. For example, it is correct within the technical skill to initiate the administration of the compound at levels lower than those required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved. However, the daily dose of the products can vary in a wide range of 0.01 to 1,000 mg per adult per day. In particular, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for symptomatic adjustment of the dose in the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, in particular from 1 mg to about 100 mg of the active ingredient. An effective amount of drug is normally supplied at a dosage level of 0.0002 mg / kg at about 20 mg / kg of body weight per day, especially from about 0.001 mg / kg to 7 mg / kg of body weight per day.

In some embodiments, the OX1R agonist described herein is administered to the subject in combination with a standard treatment. Typically, the standard treatment is selected from the group consisting of corticosteroids, immunosuppressive medications, aminosalicylates sulfasalazine, such as Mesalazine (also known as 5-aminosalicylic acid, mesalamine, or 5-ASA. Brand formulations include Apriso, Asacol, Pentasa, Mezavant , Lialda, Fivasa, Rovasa and Salofalk), Sulfasalazine (also known as Azulfidine), Balsalazide (also known as Colazal or Colazida (United Kingdom)), Olsalazine (also known as Dipentum), immunosuppressants (azathioprine, 6-mercaptopurine, methotrexate, rapamycin, cyclosporine and tacrolimus) or biological treatments such as Infliximab, Visilizumab, Adalimumab, or Vedolizumab, golimumab, tofacitinib.

The OX1R agonist described herein is typically combined with pharmaceutically acceptable excipients, and optionally sustained release matrices, such as biodegradable polymers, which will be administered in the form of a pharmaceutical composition. "Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other reaction when administered to a mammal, especially a human being, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a solid, semi-solid or non-toxic liquid filler, diluent, material encapsulator or auxiliary formulation of any kind. In the pharmaceutical compositions described herein for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active ingredient, alone or in combination with another active ingredient, can be administered in the form of unit administration, such as a mixture with conventional pharmaceutical carriers, animals and humans. Suitable forms of administration in units comprise oral forms such as tablets, gel capsules, powders, granules and suspensions or oral solutions, sublingual and oral administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal administration forms. , intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal and rectal forms of administration. Typically, pharmaceutical compositions contain vehicles that are pharmaceutically acceptable for a formulation capable of being injectable. They may be, in particular, isotonic, sterile and saline solutions (monosodium or disodium phosphate, sodium chloride, potassium, calcium or magnesium and the like or mixtures of said salts), or dry compositions, especially lyophilized, which, depending on the case, of sterilized water or physiological saline solution, allow its constitution as injectable solutions. Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for an extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that there is an easy injection capacity. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The solutions comprising the compounds described herein as free bases or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropyl cellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof and in oils. Under normal conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The antibody can be formulated in a composition in neutral or saline form. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or organic acids such as acetic, oxalic, tartaric, mandelic acids , and the like. The salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and organic bases such as isopropylamine, trimethylamine, histidine, procaine and the like. The carrier may also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof and vegetable oils. Adequate fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be caused by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions may be caused by the use in the compositions of agents that delay absorption, for example, aluminum monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active antibody in the required amount in the appropriate solvent with several of the other ingredients listed above, as necessary, followed by filtered sterilization. Generally, the dispersions are prepared by incorporating the various sterilized active ingredients in a sterile vehicle that contains the basic dispersion medium and the other required ingredients of those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and lyophilization techniques that produce a powder of the active ingredient plus any desired additional ingredient of a previously filtered sterile solution thereof. After formulation, the solutions will be administered in a manner compatible with the dosage formulation and in an amount that is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed. For parenteral administration in an aqueous solution, for example, the solution should be properly stored in a suitable buffer if necessary and the liquid diluent first turned isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this sense, the sterile aqueous media that can be employed will be known to those skilled in the art in light of the present description. For example, one dose could be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of hypopodermoclysis fluid or injected at the proposed infusion site. Some variation in the dose will necessarily occur depending on the condition of the subject being treated. The person responsible for the administration will determine, in any case, the appropriate dose for the individual subject.

The invention will be further illustrated with the following figures and examples. However, these examples and figures should not be interpreted in any way in the sense that they limit the scope of the present invention.

Figures

Figure 1: OX1R is highly expressed in inflammatory areas of patients with IBD, but not in the colonic mucosa of the controls.

Figure 2: OXA improves the ICD (weight score and colitis) of mice with DSS-induced colitis. The mice were treated orally with 5% DSS for 7 days.

Figure 3: Effects of OXA on cytokine secretion in mice with DSS-induced colitis

Figure 4: Histological appearance of the colon mucosa in mice treated with OxA (right) and untreated mice (left)

Figure 5: OXA improves the ICD (weight score and colitis) of mice with DSS-induced colitis. Mice were treated orally with 5% DSS for 7 days in the presence or absence of daily intraperitoneal injection of OxA (0.22 pmoles / kg). The ICD score, including weight loss, the severity of diarrhea, the presence of blood in the stool, inflammation and edema were estimated using a scale ranging from 0 (no symptoms) to 4 (severe colitis). DSS, dextran sodium sulfate; WT, natural mice.

Figure 6: Effects of OXA on cytokine secretion in EXCY2 mice. Colones of EXCY2 mice in untreated mice (white) and treated with OxA treatment (0.22 pmoles / kg) (black) were resected after animal sacrifice. Then, protein extraction was performed by tissue disruption. Cytokines were determined using CBA cytokine kits (see Materials and Methods).

Figure 7: OXA further improves the ICD (weight and colitis score) of EXCY2 mice than the standard treatment of 5-aminosalicylic acid in gold (5-ASA). Mice were injected intraperitoneally or not with OxA (0.22 pmol / kg) or treated orally with 5-ASA (0.053 g / kg). The ICD score that includes weight loss, severity of diarrhea, presence of blood in the stool, inflammation and edema was calculated using a scale ranging from 0 (no symptoms) to 4 (severe colitis).

Examples

Example 1

The inventors have shown that OX1R was expressed in human inflammatory bowel disease (IBD), including Crohn's disease and UC. In fact, the use of specific antibodies directed against OX1R in the immunohistochemical experiment (IHC) of approximately 40 inflamed colon samples revealed the ectopic presence of OX1R in both epithelial and immune cells (Figure 1). In contrast, OX1R was not expressed in normal colonic mucosa (Figure 1).

Based on these observations, the effect of Orexin A (OxA) on acute inflammation in mice treated with Sodium Dextran Sulfate (DSS) has been investigated. OxA was preferably chosen in these experiments since the presence of two disulfide bridges gave it more stability. DSS induces acute colitis characterized by weight loss, bloody diarrhea, intestinal ulcerations and granulocyte infiltration. The results of the inventors indicate that treatment with OxA in mice treated with DSS orally improves the disease activity index (DAI) evaluated by measuring the weight, length of the colon, diarrhea and the presence of blood in the stool (Figure 2).

These observations are confirmed by the histological aspect of the colon epithelium (histological score by a gastrointestinal pathologist). Therefore, OxA probably has an anti-inflammatory effect on DSS-induced colitis. To confirm this hypothesis, the anti-inflammatory effect of OxA treatment on cytokine secretion in colon extracts of mice treated with DSS has been investigated. Analysis of the cytokine profile revealed that OxA reduces the secretion of "proinflammatory" cytokines as homologues of TNFa, IL6, IL8 and ILlB in colon extracts of mice with DSS-induced colitis (Figure 3). In contrast, OxA has no effect on the secretion of cytokines INFy, IL10 and ILl2 in colon extracts (Figure 3).

The DSS mouse model has been widely used to decipher mucosal inflammation in IBD, the pathogenesis of UC and preclinical studies. However, mice show differential susceptibilities and responsiveness to DSS-induced colitis depending on their genetic origin, gender. The DSS model is a faithful model of inflammation, but part of ulcerative colitis, partly due to the lack of the inflammation gradient from the rectum to the proximal colon that is usually observed in UC, any deregulation of ER stress in goblet cells It is known to be an early etiologic factor of UC, and without digestive or extradigestive complications of UC such as cholangitis and colorectal cancer, respectively. The use of the model of genetically modified mice represents a good alternative to the model of mice with chemically induced colitis. The recent development of a new model of spontaneous mouse type UC (EXCY2 mice, document WO / 2012/140516) consisting of a double KO of IL10 and Nox1 (IL10 - / -; Nox1 '/ _) is ideal for studying the effect of OxA on spontaneous colitis. In fact, EXCY21) mice develop a spontaneous colitis at 6/7 weeks of age with an ascending gradient from the rectum and reproduce all the molecular characteristics observed in UC including loss of goblet cells, ER stress deregulation, protective effect of tobacco; 2) spontaneous colon cancer at 8 months of age (35-40% of the mice); 3) development of mild cholangitis.

The results of the inventors indicate that treatment with OxA (1 pM OxA, 2 IP / week for 3 weeks) relieved severe colitis in EXCY2 mice 10 weeks old, EXCY2 mice treated with OxA exhibited a normal colonic mucosa (appearance General crypt size, crypt size, presence of goblet cells, absence of immune cell infiltration) compared to vehicle-treated EXCY2 mice (Figure 4). Interestingly, it was shown that OxA / OXlR induced two major deregulated pathways involved in the onset of colitis (oxidative stress and ER stress) in both EXCY2 mice and Caco2 cells.

In conclusion, these data indicate that 1) OxA could show original anti-inflammatory properties in the mouse model treated with DSS; 2) OxA strongly protects against the spontaneous colitis developed in the EXCY2 mouse model and triggers mucosal healing, certainly, by controlling the different pathways involved in the onset of UC. Taking this proof of concept into account, the orexin / OXIR system represents an innovative and effective goal in the treatment of inflammatory bowel diseases, particularly in ulcerative colitis.

Example 2

OXA improves the ICD (weight score and colitis) of mice with DSS-induced colitis.

As shown in Figure 5, when mice with DSS-induced colitis were treated with OxA, the DAI score improved (approximately 1.5).

Effects of OXA on cytokine secretion in EXCY2 mice. The data of the inventors indicate that treatment with OxA (0.22 pmoles / kg OxA, 2 IP / week for 3 weeks) relieved severe colitis in EXCY2 mice 10 weeks old. The cytokine pattern of EXCY2 mice treated with OxA showed a strong reduction in the secretion of proinflammatory cytokines including TNFa, IL6, IFN ^and , MCP1 and IL12 (Figure 6). In contrast, the secretion of anti-inflammatory cytokines IL10 did not change (Figure 6). These results indicated that OxA has a strong effect on the inflammatory response.

OXA improves the DAI (weight and colitis score) of EXCY2 mice better than the standard gold treatment of 5-aminosalicylic acid (5-ASA). As shown in Figure 7, OxA treatment of EXCY2 mice showed a DAI score of 1.5 compared to untreated EXCY2 mice that had a DAI score of 4 corresponding to severe colitis. Treatment of EXCY2 mice with 5-ASA reduced the DAI score compared to untreated mice, that is, 3 versus 4, respectively. These data clearly demonstrated that OxA was more efficient than the standard 5-ASA gold treatment in symptoms developed in EXCY2 mice.

References

Throughout this application, various references describe the state of the art to which this invention pertains.

Claims (5)

1. At least one OX1R agonist for use in a method of treating an inflammatory bowel disease in a subject in need, in which the OX1R agonist is a polypeptide that has at least 70% identity with SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4. 2. At least one OX1R agonist for use according to claim 1, wherein the inflammatory bowel disease is ulcerative colitis, Crohn's disease, especially Crohn's disease in a state that specifically affects the colon with or without ileitis, colitis microscopic, infectious colitis caused by bacteria or viruses, radiation colitis, ischemic colitis, pediatric colitis, indeterminate colitis and functional intestinal disorders without obvious anatomical abnormalities. 3. At least one OX1R agonist for use according to claim 1, wherein the OX1R agonist is a polypeptide selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 . 4. At least one OX1R agonist for use according to claim 1, wherein the OX1R agonist is an immunoadhesin comprising a polypeptide characterized by SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 merged to an Fc domain. 5. At least one OX1R agonist for use according to claim 1, wherein the OX1R agonist is administered to the subject in combination with a standard treatment selected from the group consisting of corticosteroids, immunosuppressive drugs, aminosalicylates sulfasalazine, such such as Mesalazine, Sulfasalazine, Balsalazide, Olsalazine, immunosuppressants such as azathioprine, 6-mercaptopurine, methotrexate, rapamycin, cyclosporine and tacrolimus or biological treatments such as Infliximab, Visilizumab, Adalimumab or Vedolizumab, golimimibumab, golacimibumab, golacimibumab.